Project Abstract This application seeks to define the molecular mechanisms and fitness benefits of alginate overproduction by mucoid variants of Pseudomonas aeruginosa. Specifically we will define features of the alginate biofilm matrix that enhance P. aeruginosa persistence and better understand the role of alginate in maintaining passive protection to the P. aeruginosa microbial community in the CF airway. This proposal will use state-of-the art molecular, biochemical, immunological, and genetic approaches to probe aspects of alginate pathogenesis and to reevaluate some long-standing paradigms. To date, the mechanisms underlying biofilm formation by alginate producing bacteria is not well understood. Aim 1 will investigate the biochemical and genetic basis for alginate biofilm matrix maintenance of biofilm integrity, structure, and antimicrobial resistance properties. While it is clear that mucoid variants predominate during chronic airway infection in CF patients, rarely are they present as pure clonal mucoid isolates. Instead, mucoid bacteria are typically associated with mixed populations of wild type P. aeruginosa that have not undergone mucoid conversion, as well as spontaneous suppressors that arise. The goal of Aim 2 is to test the hypothesis that this mixed consortia confers fitness benefits to the P. aeruginosa community as a whole that is not observed in either nonmucoid or mucoid bacteria alone. Alginate expression in the CF lung correlates with a poor clinical prognosis. However there are gaps in our knowledge regarding specific properties that alginate overproduction affords bacteria and the microbial consortia within the airway. The completion of these aims will lead to a deeper understanding of alginate biology and translate into novel therapies or interventions for CF patients colonized with mucoid P. aeruginosa.